Dynamics and organization of alpha3-containing glycine receptors using single molecule detection

Abstract

The Glycine receptor (GlyR) is a chloride permeable ligand gated ion channel and that can mediate synaptic inhibition. Due to a possible involvement in the pathophysiology of temporal lobe epilepsy,the different properties of GlyRs containing alpha3L and K subunit isoforms are currently investigated. Previous characterizaions of homomeric receptors consisting of these isoforms have shown a difference in their electrophysiological properties and their membrane distribution as observed by diffraction-limited fluorescence microscopy. We studied these isoforms, when separately expressed in tranfected HEK 293T cells, by using single molecule tracking (SMT) in living cells and direct stochastic optical reconstruction microscopy (dSTORM) on fixated cells. For both techniquest the GlyRs are stained using a primary antibody directly labeled with Alexa Fluor dyes. The dSTORM experiments support the observation that the alpha3L GlyR are clustered, while the alpha3K GlyRs are more uniformly spread. The analysis of the short range diffusion coeffcients obtained by SMT reveals the presence of heterogenous motion for both isoforms. The K-isoform has a higher fraction of fast diffusion. In contrast, the L-isoform is more associated with slow diffusion and appears to undergo hindered diffusion.